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ResearchCite this article: Cha J, Sun X, Bartos A,

Fenelon J, Lefevre P, Daikoku T, Shaw G,

Maxson R, Murphy BD, Renfree MB, Dey SK.

2013 A new role for muscle segment homeobox

genes in mammalian embryonic diapause.

Open Biol 3: 130035.

http://dx.doi.org/10.1098/rsob.130035

Received: 22 February 2013

Accepted: 2 April 2013

Subject Area:developmental biology/cellular biology/

genetics/molecular biology

Keywords:MSX genes, delayed implantation, embryonic

diapause, mouse, mink, tammar wallaby

Author for correspondence:Sudhansu K. Dey

e-mail: sk.dey@cchmc.org

Electronic supplementary material is available

at http://dx.doi.org/10.1098/rsob.130035

& 2013 The Authors. Published by the Royal Society under the terms of the Creative Commons AttributionLicense http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the originalauthor and source are credited.

A new role for muscle segmenthomeobox genes in mammalianembryonic diapauseJeeyeon Cha1, Xiaofei Sun1, Amanda Bartos1, Jane Fenelon2,3,

Pavine Lefevre2, Takiko Daikoku1, Geoff Shaw3, Robert Maxson4,

Bruce D. Murphy2, Marilyn B. Renfree3 and Sudhansu K. Dey1

1Division of Reproductive Sciences, Cincinnati Children’s Research Foundation, Cincinnati,OH 45229, USA2Center for Animal Reproduction, Faculty of Veterinary Medicine, University of Montreal,Saint Hyacinthe, Quebec, Canada J2S 7C63Department of Zoology, University of Melbourne, Melbourne, Victoria 3010, Australia4Department of Biochemistry and Molecular Biology, Keck School of Medicine,Norris Comprehensive Cancer Center and Hospital, University of Southern California,Los Angeles, CA 90033, USA

1. SummaryMammalian embryonic diapause is a phenomenon defined by the temporary

arrest in blastocyst growth and metabolic activity within the uterus which syn-

chronously becomes quiescent to blastocyst activation and implantation. This

reproductive strategy temporally uncouples conception from parturition until

environmental or maternal conditions are favourable for the survival of the

mother and newborn. The underlying molecular mechanism by which the

uterus and embryo temporarily achieve quiescence, maintain blastocyst survi-

val and then resume blastocyst activation with subsequent implantation

remains unknown. Here, we show that uterine expression of Msx1 or Msx2,

members of an ancient, highly conserved homeobox gene family, persists in

three unrelated mammalian species during diapause, followed by rapid down-

regulation with blastocyst activation and implantation. Mice with uterine

inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation.

Remarkably, the North American mink and Australian tammar wallaby share

similar expression patterns of MSX1 or MSX2 as in mice—it persists during

diapause and is rapidly downregulated upon blastocyst activation and implan-

tation. Evidence from mouse studies suggests that the effects of Msx genes in

diapause are mediated through Wnt5a, a known transcriptional target of uter-

ine Msx. These studies provide strong evidence that the Msx gene family

constitutes a common conserved molecular mediator in the uterus during

embryonic diapause to improve female reproductive fitness.

2. IntroductionEmbryonic diapause is a transient state in which embryos at the blastocyst stage

are arrested in growth and metabolic activity in synchrony with uterine quies-

cence. This provisional delay of implantation allows pregnancy to withstand

harsh environmental conditions, or adapt to a changing photoperiod or mater-

nally derived stimuli such as lactation, thereby allowing pregnancy to resume

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during conditions favourable to rear young [1–4]. This repro-

ductive strategy is widespread, occurring in nearly 100

mammalian species across seven orders [1,2].

Although features of diapause vary across species with

respect to endocrine status [1,2,4], the presumed purpose is to

prolong the gestational period, allowing young to be born

at optimal times of the year with respect to a species’ nutritio-

nal and environmental needs and apportioning the mother’s

metabolic resources to nurse sequential litters. The pervasive

existence of mammalian embryonic diapause implies that the

pressure to safeguard and preserve procreation under diverse

conditions through sexual reproduction is one of the fundamen-

tal forces in mammalian evolution. However, the underlying

molecular mechanism by which the uterus and embryo tem-

porarily achieve growth restriction and then resume blastocyst

activation for implantation under favourable conditions

remains largely unknown.

Delayed implantation can occur naturally during lactation

after postpartum mating (facultative delay). The embryo devel-

ops to the blastocyst stage and remains dormant within the

quiescent uterus, while the newly born pups are suckling,

allowing sufficient time to nurse sequential litters. In mice,

this stems from heightened levels of pituitary prolactin from

the suckling stimulus that attenuates ovarian oestrogen

secretion [5,6]; removing this stimulus can trigger blastocyst

reactivation (implantation competency) and implantation.

This strategy is also present in several diapausing mammals,

including the Australian tammar wallaby [3]. In this case, pro-

lactin is luteostatic, rendering the corpus luteum quiescent and

thus inhibiting progesterone (P4) secretion required for uterine

secretory activity and reactivation. Removal of the suckling

pouch young after the summer solstice induces reactivation.

Tammars also display a seasonal diapause (obligatory)

controlled by photoperiod and melatonin secretion after the

winter solstice [3]. Mink, ferret and skunk, among others,

also have seasonal diapause, implicating day length and mela-

tonin levels in regulating diapause via prolactin [2]. Irrespective

of the type of delay, embryonic diapause is under maternal

regulation in all species studied to date. Although the endo-

crine milieu behind various natural inducers has been

characterized in several diapausing species [1,2,4], the molecu-

lar mechanism by which it promotes synchronized uterine

quiescence and blastocyst dormancy, while maintaining

implantation competency is poorly understood.

Our recent studies in mice show that muscle segment homeobox(Msx) genes play critical roles in preparing the uterus to the

receptive state for blastocyst implantation during normal preg-

nancy. The expression is transient, appearing on the morning

of day 4 (the day of uterine receptivity) and then being dramati-

cally downregulated approaching implantation and thereafter

[7]. By contrast, we show here that Msx1 is highly expressed in

the mouse uterine epithelium during embryonic diapause

induced by various approaches and becomes undetectable

with the initiation of implantation. More importantly, we also

found that mice with conditional uterine inactivation of Msx(Msx1 and Msx2) have drastically reduced blastocyst recovery

and survival. These results suggested that Msx genes are critical

for conferring true dormancy and implantation competence.

These intriguing results led us to study the role of Msx genes

in embryonic diapause and delayed implantation in representa-

tive species of three mammalian orders: mice (Eutheria:

Rodentia), American mink (Eutheria: Carnivora) and Australian

tammar wallabies (Marsupialia: Diprotodontia). We observed

that Msx genes share this conserved role in unrelated diapausing

mammals. MSX1 expression persists in the luminal and glandu-

lar epithelia of American mink (Neovison vison) under obligate

delay regulated by photoperiod. The expression is downregu-

lated with the resumption of blastocyst activation and

implantation. Similarly, another family member, MSX2 [8],

remains highly expressed in the uterus of the Australian

tammar wallaby (Macropus eugenii) undergoing lactational dia-

pause, but again is downregulated upon exit from diapause

and is absent after blastocyst attachment. As expression of Msxgenes persists during the delay in these different species, and

because mice with uterine deletion of Msx genes fail to undergo

true delay, we propose that Msx genes constitute a gene family

whose function is conserved not only for normal implanta-

tion, but is also for the maternal regulation of mammalian

embryonic diapause.

3. Material and methods3.1. Animals

3.1.1. Mice

Mice with uterine deletion of Msx1 and Msx2 (Msx1/Msx2loxP/loxP

PgrCre/þ ¼Msx1/Msx2d/d) and control littermates (Msx1/Msx2loxP/loxP Pgrþ/þ ¼Msx1/Msx2f/f ) were generated as

previously described [7]. Msx1LacZ, Lif2/2 and Pgr-Cre mice

were initially provided by Robert Benoit, Phillippe Brulet,

and John Lydon and Francesco DeMayo, respectively

[7,9,10]. All mice used in this investigation were housed in

barrier facilities in the Cincinnati Children’s Hospital Medical

Center’s Animal Care Facility according to National Institutes

of Health and institutional guidelines for the use of labora-

tory animals. All protocols for this study were reviewed

and approved by the Cincinnati Children’s Research Foun-

dation’s Institutional Animal Care and Use Committee.

Mice were provided with autoclaved rodent LabDiet 5010

(Purina) and UV-light-sterilized RO/DI (reverse osmosis/

deionized) constant circulation water ad libitum.

3.1.2. Mink

All procedures involving live animals were approved by the

University of Montreal Animal Care Committee, which is

accredited by the Canadian Council on Animal Care. Investi-

gations were carried out during two consecutive annual

breeding seasons in 2011 and 2012 using ranch mink of the

Pastel variety acquired from a local farm (A. Richard, Saint

Damase, Quebec, Canada). During the mink breeding season,

which begins in early March, each female was bred to two fer-

tile males according to usual farm mating practices. Uteri were

collected in the delayed state, 7–9 days after the final mating,

and diapause confirmed by the occurrence of blastocysts in

the reproductive tract [11]. In a subset of animals, embryo reac-

tivation was synchronized by daily injection of 1 mg kg21 per

day ovine prolactin (Sigma-Aldrich, Oakville, Ontario,

Canada) [12] beginning on 21 March and continuing for the fol-

lowing 10 days. The first day of prolactin injection was

designated day 0 of embryo reactivation. Uterine horns were

collected at days 0 (diapause), 9 and 14 after prolactin-induced

reactivation. Implantation occurred approximately 12 days

after initiation of prolactin treatment, and uteri with implan-

tation sites were collected at day 14. Tissues were snap-frozen

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and stored at 2808C until they were shipped to Cincinnati

Children’s Research Foundation on dry ice for in situhybridization and immunofluorescence analyses.

3.1.3. Tammar wallaby

Tammar wallabies (M. eugenii) of Kangaroo Island origin

were kept in open grassy yards in a breeding colony main-

tained by the University of Melbourne. Their diet was

supplemented with fresh fruit, vegetables and lucerne

cubes, and water was supplied ad libitum. Care and treat-

ment of animals conformed to the National Health and

Medical Research Council Australian 2004 guidelines. All

animal handling and experimentation were approved

by the University of Melbourne Animal Experimentation

Ethics Committees. Tammar uterine samples were obtained

as previously described [13]. Before and during embryonic

diapause, the stage of pregnancy was based on the postpar-

tum age of the pouch young (day 0–day 350 postpartum).

Females were checked regularly for births, and if the date

of birth was not known, then the ages of pouch young

were extrapolated from growth curves based on head

length measurements [14]. In this study, adult females with

a pouch young older than day 8 postpartum were presumed

to be carrying a diapausing blastocyst [3]. Before reactivation

from diapause can occur, the pouch young-induced luteal

inhibition must be removed for three consecutive days

[15,16]. Therefore, after diapause, the reactivation stages of

pregnancy were determined relative to the day of the removal

of the pouch young (days after RPY). All tissues were

collected under RNase-free conditions. Snap-frozen tissues

were stored at 2808C, and shipped via cryoshipper in

liquid N2 to Cincinnati Children’s Research Foundation for

in situ hybridization, immunostaining and qPCR.

3.1.4. Analysis of delayed implantation and activation in mice

Littermate Msx1/Msx2f/f and Msx1/Msx2d/d females were

mated with wild-type (WT) fertile males to induce pregnancy.

To induce delayed implantation, mice were ovariectomized on

day 4 of pregnancy and administered progesterone (P4, 2 mg

per dose, sc) in sesame oil until day 8 of pregnancy (4 days

of dormancy) or day 10 (6 days of dormancy). Anti-oestrogen

(ICI 182,780, Zeneca Pharmaceuticals, 50 mg 100 ml21 dose, sc)

was administered on day 3 at 1800 h and day 4 between 0800

and 0900 h to induce delay. Uteri were flushed with physio-

logical saline to recover blastocysts. For activation, mice were

injected either with recombinant leukaemia inhibitory factor

(LIF) (50 mg per mouse, ip) on day 6 at 1000 and 1600 h or

with 25 ng oestradiol-17b (E2). Implantation sites were exam-

ined 24 or 48 h later. Implantation sites were visualized by

intravenous injection of a Chicago blue dye solution, and

implantation sites, demarcated by distinct blue bands, were

collected. To study lactational delay, pregnant females were

housed with males on day 17 of pregnancy onwards and

checked for vaginal plugs on days 19–20 [5,6,17]. Upon deliv-

ery (P1), pups were either removed or kept with the mother to

suckle for 6 days, and uteri were collected 6 days postpartum

(P6) for in situ hybridization. Delay in implantation during lac-

tation depends on the intensity of suckling stimuli; there is

often failure of delay if only a few pups are allowed to

suckle [18]. Therefore, only dams with six to eight pups per

litter were used in this study.

3.2. Hormone treatmentNon-pregnant WT or Msx1LacZ female mice were ovari-

ectomized and allowed to recover for 10 days. Females

were administered subcutaneously 100 ml of sesame oil,

2 mg P4, 25 ng E2, or P4 þ E2, and uteri were collected 24 h

later and snap-frozen for in situ hybridization. Tissues from

each treatment were processed onto the same slide. Litter-

mate Msx1/Msx2f/f and Msx1/Msx2d/d females were

administered 2 mg P4 on day 3 and day 4 of pregnancy.

The morning of finding the vaginal plug was considered

day 1 of pregnancy. Implantation sites were visualized by

intravenous injection of a Chicago blue dye solution, and

implantation sites were counted [19,20]. If no sites were

found, then uteri were flushed with physiological saline to

recover blastocysts to confirm pregnancy.

3.3. ImmunostainingImmunohistochemistry on mouse tissues was performed in

formalin-fixed, paraffin-embedded sections using specific

antibodies. Primary antibodies for cyclooxygenase-2 (Cox2)

(laboratory generated, 1 : 200), cytokeratin 8 (Developmental

Studies Hybridoma Bank, 1 : 200), Msx1/2 (recognizes Msx1

and Msx2, Developmental Studies Hybridoma Bank, 1 : 50),

Msx1 (Santa Cruz, 1 : 200), phospho-histone H3 (Santa Cruz,

1 : 200) and Ki67 (Neomarkers, 1 : 300) were used. In tammar

wallaby studies, tissues were fixed in 4 per cent paraformalde-

hyde and paraffin-embedded. To compare intensity of

immunostaining between tissues, tissue sections from both

genotypes on specific day of pregnancy or differential stages

of diapause and reactivation were processed on the same

slide. A Histostain-Plus (diaminobenzidine) kit (Invitrogen)

was used to visualize specific antigens. Immunofluorescence

for Msx1 on mink tissues was performed using Msx1 antibody

(Santa Cruz), and secondary antibodies Cy2 conjugated

donkey anti-rabbit (Jackson Immuno Research) on frozen sec-

tions. Nuclear staining was performed using DAPI.

Immunofluorescence was visualized with confocal microscopy

(Nikon Eclipse TE2000).

3.4. In situ hybridizationIn situ hybridization was performed as previously described

[21]. Uterine samples from mouse, mink and tammar wallaby

were collected and snap-frozen. Uterine sections (12 mm

thickness) were mounted onto poly-L-lysine-coated slides,

fixed in cold 4 per cent paraformaldehyde, acetylated and

hybridized at 458C for 4 h in formamide hybridization

buffer containing species-specific 35S-labelled cRNA probes.

RNase A-resistant hybrids were detected by autoradiography

with Kodak NTB-2 liquid emulsion. Parallel sections were

hybridized with the corresponding sense probes. To compare

mRNA patterns and intensities, sections of uteri from control

and experimental groups or from different days of diapause

and reactivation were placed onto the same slide and pro-

cessed for hybridization. Mouse-specific cRNA probes for

Msx1, Hoxa10, Ihh, Ptgs2, Bmp2 and Wnt5a were used for

hybridization. For mink and tammar wallaby experiments,

dog-specific cRNA probes for MSX1 and WNT5A and

tammar wallaby-specific cRNA probe for MSX2, respectively,

were generated from published genomic sequences.

ovariectomized P4-treated delay lactational delay

day1

vaginalplug

P6

+

pupsremoved

postpartum (day 6)

plug

(+

)no

t suc

klin

gpl

ug (

+)

suck

ling

plug

(–)

suck

ling

+

delivery(P1)

day 17± male ± pups

tissuecollection

anti-oestrogen treated delay

delayed

(a) (c)

(b)

Msx

1

Msx1

Msx

1

P4 P4 + E2

activated

delayed

ICI 182,780 ICI + LIF (24 h)

bl

ge

le

bl

bl

ICI + LIF (48 h)

activated

Figure 1. Persistent uterine expression of Msx1 under different inducers of delayed implantation in mice. (a) Ovariectomized, delayed implanting mice treated withP4 or P4 þ E2 were killed on day 8 of pregnancy. In situ hybridization showed distinct Msx1 expression in luminal and glandular epithelia during delay with rapiddownregulation following attachment reaction and embryonic activation 24 h after an E2 injection. (b) Oestrogen receptor antagonist (ICI 182,780) injected on days 3and 4 induced delay, and LIF was injected twice on day 6 to terminate delay. In situ hybridization of uterine sections examined 24 or 48 h later showed persistentMsx1 expression in delayed conditions which became undetectable with implantation initiation. (c) Females on day 17 of pregnancy were housed with males andchecked for vaginal plug on days 19 – 20. Upon delivery (P1), pups were either removed (white arrow) or kept with the mother; uteri were collected six dayspostpartum (P6). Delayed implanting, lactating mothers had high uterine epithelial Msx1 expression, which became undetectable following implantation uponwithdrawal of suckling stimulus. Arrowheads denote embryos (bl). ge, glandular epithelium; le, luminal epithelium. Scale bar, 500 mm.

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3.5. RT-PCR and qPCRIn mink samples, the housekeeping gene GAPDH (encoding

glyceraldehyde-3-phosphate dehydrogenase) was used as an

internal control. qPCR was performed using primers: 50-

AAGTTCCGCCAGAAGCAGTA-30 and 50-AGCCATCTT-

CAGCTTTTCCA-30 for MSX1 (annealing temperature 688C,

product size 220 bp); 50-AAAGGCAGTGACCTGTT-30 and 50-

GGTGGGGCTCATATGTCT-30 for MSX2 (expected product

size 381 bp); and 50-TCCCCACCCCCAATGTG-30 and 50-

CCCTCTGATGCCTGCTTCA-30 for GAPDH. In tammar wal-

laby samples, the housekeeping gene ACTB (encodes b-actin)

was used as an internal control, and RT-PCR was performed at

35 cycles. RT-PCR and qPCR were performed using primers:

50-TTG CTG ACA GGA TGC AGA AG-30 and 50-AAA GCC

ATG CCA ATC TCA TC-30 for tammar wallaby-specific ACTB(annealing temperature 608C; product size 247 bp); 50-

CCGAAAGTCCTGAGAAGCAG-30 and 50-GAGGCTCAGA

GAGCTGGAGA-30 for wallaby-specific MSX1 (expected pro-

duct size 233 bp); and 50-TCGCAGGGTCAAAGTGTCTAG-30

and 50-GTTGTGGGACTCAAGTGTCTT-30 for wallaby-specific

MSX2 (annealing temperature 638C; product size 311 bp).

Primers were generated from the published tammar wallaby

sequence and dog sequence.

3.6. StatisticsStatistical analyses were performed, using two-tailed Student’s

t-test. A value of p , 0.05 was considered statistically significant.

4. Results4.1. Msx1 expression persists in the mouse uterine

epithelium during delayed implantation inducedby multiple approaches

Ovariectomy of pregnant female mice prior to preimplantation

oestrogen secretion on day 4 induces delayed implantation,

and blastocyst dormancy can be maintained for several days

to weeks with continued P4 treatment [22–24]. Blastocyst reac-

tivation and implantation will not occur unless initiated with

an injection of E2 or its downstream target LIF [5]. Msx1expression persisted in these delayed implanting females

beyond its tightly regulated, transient expression on day 4

followed by rapid downregulation approaching attachment

reaction upon activation by E2 in a P4-primed uterus

(figure 1a).

We next addressed whether the expression of Msx1 persists

in delayed uteri induced by different approaches. Delayed

implantation was induced in WT females by administration

of a specific anti-oestrogen (ICI 182,780) on day 3 afternoon

prior to preimplantation ovarian oestrogen secretion on day

4 morning to block oestrogen action; they were then killed

on day 8. We found that under this condition Msx1 expression

persisted in the luminal and glandular epithelium (figure 1b).

To determine whether LIF supplementation would initiate

reactivation of dormant blastocysts and implantation, LIF

(50 mg per mouse) was intraperitoneally injected at 1000 and

day 8 (4 days of dormancy)

day 10 (6 days of dormancy)

Msx1/Msx2 f/f Msx1/Msx2 d/d

Msx1/Msx2 f/f

Msx1/Msx2 f/f d/d

Msx1/Msx2 f/f d/d

6

5

4

3

2

1

0

(20/20)

no. r

ecov

ered

bla

stoc

ysts

(mea

n±

s.e.

m.)

no. r

ecov

ered

bla

stoc

ysts

(mea

n±

s.e.

m.)

(21/21)

p=0.004

8

6

4

2

0

(7/8)

(5/19)

p=0.015Msx1/Msx2 d/d

(a) (b)

(c) (d)

Figure 2. Uterine deletion of Msx genes fails to manifest true delay with reduced embryo survival. (a) Number of dormant blastocysts recovered from ovari-ectomized, P4-treated Msx1/Msx2d/d females was significantly lower compared with floxed littermates (Msx1/Msx2 f/f ) on day 8 under delayed conditions.Several blastocysts recovered from deleted females showed signs of degeneration and poor morphological appearance compared with those recovered fromfloxed littermates. (b) A further reduction in number of recovered blastocysts was observed in deleted females when delay was extended to 10 days of pregnancy,and in some blastocysts, luminal epithelial cells were attached to the abembryonic trophectoderm (blue arrows). A subset of P4-treated, Msx1/Msx2d/d uteri showedsmall swellings with faint blue bands at the site of blastocysts on day 10 ( pseudo-implantation sites, arrowheads).

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1600 h on day 7 in these anti-oestrogen-treated delayed

implanting mice. LIF initiated implantation in these anti-

oestrogen-treated delayed mice. Implantation sites were then

examined for Msx1 expression 24 and 48 h later by in situhybridization. Strikingly, epithelial Msx1 expression was

progressively downregulated from the implantation site by

24 h to undetectable levels by 48 h (figure 1b).

Finally, we explored whether uterine Msx1 expression

persists in mice with lactational delay, a natural delay

induced by suckling pups following postpartum mating;

removal of the suckling stimulus promptly initiates embryo-

nic activation and implantation. Pregnant females were

housed with fertile males just prior to parturition. Mating

was confirmed by the presence of a vaginal plug, and

suckling was allowed to continue for 6 days in plug-positive

females. We found that these lactating mice showed delayed

implantation with unimplanted dormant blastocysts

recovered from their uteri. Again, Msx1 was distinctly

expressed in the epithelium of these delayed uteri. More

importantly, uterine Msx1 expression became undetectable

in those postpartum mated females from which pups were

removed following delivery (figure 1c). Collectively, delayed

implantation conferred by surgical (ovariectomy), pharma-

cological (anti-oestrogen) or natural (lactation) approaches

consistently exhibited heightened uterine Msx1 expression

with rapid downregulation upon blastocyst activation and

implantation.

4.2. Mice with uterine inactivation of Msx genes fail toachieve true delay and show compromisedblastocyst survival

Delayed implantation occurs only if blastocyst dormancy is syn-

chronized with uterine quiescence, and conversely, implantation

can resume if blastocyst activation is superimposed on uterine

receptivity. The persistent uterine Msx1 expression during

delay and its downregulation following blastocyst activation

with initiation of implantation suggested that Msx1 is critical

for synchronized quiescence of both the uterus and blastocyst.

Although Msx1 is primarily expressed in the uterus, Msx2 is

expressed as a compensatory partner in a similar manner as

Msx1 in the uterus lacking Msx1 [7]. To obtain genetic evidence

whether uterine Msx1 and Msx2 influence embryonic diapause

under delayed conditions, we used mice with uterine inacti-

vation of both Msx1 and Msx2 (Msx1/Msx2d/d) achieved by

Cre recombinase expression driven by the progesterone receptor

promoter. We found substantially fewer dormant blastocysts in

ovariectomized, P4-primed Msx1/Msx2d/d females on day 8

(4 days of dormancy) compared with littermate Msx1/Msx2f/f

(control) females under similar treatment conditions; many

blastocysts recovered from Msx1/Msx2d/d uteri showed signs

of abnormal morphology and degeneration. Thus, uteri lacking

Msx genes subject to dormancy conditions are not conducive

to blastocyst survival (figure 2a,b). When the delay was extended

Ptgs2

day 10 (6 days of dormancy)

Bmp2 CK8

Msx

1/M

sx2f/

f

uter

usM

sx1/

Msx

2d/d

pseu

do-I

Sf/

fd/

dW

nt5a

Msx1/Msx2 f/f

blastocysts(6 days of dormancy)

day 10 (6 days of dormancy)uterus uterus pseudo-IS

M

AM

Msx1/Msx2 d/d

no. c

ells

/bla

stoc

yst

(mea

n±

s.e.

m.)

Msx1/Msx2 f/f d/d

p=0.00003

n=16120

80

40

n=12

(a)

(b) (c)

(d)

Figure 3. Blastocysts in Msx1/Msx2d/d females under delayed conditions showimplantation-like responses ( pseudo-implantation) without epithelial invasionand display increased cell proliferation. (a) Pseudo-implantation sites ( pseudo-IS) in Msx1/Msx2d/d females on day 10 under dormant conditions show increasedepithelial and stromal Ptgs2 expression, and stromal Bmp2 at the site of blastocystwithout sign of trophectoderm invasion through the intact epithelial barrier asdemarcated by cytokeratin 8 (CK8) staining. Arrowheads denote blastocysts.Scale bar, 250 mm. (b,c) Blastocysts recovered from Msx1/Msx2d/d femalesappeared larger with higher cell numbers compared with those recovered fromMsx1/Msx2f/f delayed uteri. Blue, nuclear staining. Scale bar, 50 mm. (d ) P4-trea-ted delayed implanting Msx1/Msx2d/d uteri and pseudo-implantation sitesshowed increased uterine Wnt5a expression, particularly at the site of blastocyst,compared with floxed littermates. Arrowheads denote blastocysts. Scale bar,125 mm. M, mesometrial pole; AM, anti-mesometrial pole.

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to 6 days (day 10 of pregnancy), blastocyst recovery was further

reduced. In addition, several females examined yielded no blas-

tocysts (figure 2c,d and electronic supplementary material, table

S1). The initiation of blastocyst attachment can be detected by an

intravenous injection of a blue dye, because there is increased

endometrial vascular permeability at the site of attachment

[19,20]. Intriguingly, under ovariectomized, P4-treated delayed

conditions, a small number of Msx1/Msx2d/d females (33%)

showed faint blue bands with small swellings. These swellings

were not due to incomplete ovariectomy, as their size was remi-

niscent of day 5 or 6 of normal pregnancy, not day 10 (figure 2cand electronic supplementary material, figure S1a,b). Blastocysts

were intimately apposed to the luminal epithelium with some

recovered blastocysts having epithelial cells adhered to the tro-

phectoderm (figure 2c, blue arrows). We also found some signs

of decidualization (stromal cell proliferation) at the site of blasto-

cyst with induction of Ptgs2 (Cox2) expression, an early marker of

vascular permeability and decidualization, similar to that seen

on day 5 of normal pregnancy (figure 3a and electronic sup-

plementary material, figure S1c) [21]. Bmp2, critical for

decidualization [19,25], was also expressed at the same site,

again reminiscent of expression at the implantation site on day

5 of normal pregnancy (figure 3a and electronic supplementary

material, figure S1c). However, the luminal epithelium in Msx-

deleted uteri was intact as shown by cytokeratin staining with

no signs of trophectoderm invasion (pseudo-implantation;

figure 3a). By contrast, delayed Msx1/Msx2f/f females showed

features of true dormancy with absence of blue bands, no decid-

ual swellings and undetectable levels of Bmp2 and Ptgs2 at the

site of blastocyst; an expected number of dormant blastocysts

were recovered from uteri of these mice. As anticipated, Msx1/Msx2d/d uteri fail to resume implantation by an E2 injection in

contrast to floxed mice (see the electronic supplementary

material, figure S2 and table S2). Collectively, the findings

provide genetic evidence that Msx is critical for regulating the

uterine milieu to confer uterine quiescence and blastocyst

dormancy and viability.

It is known that dormant blastocysts exhibit little or no

mitotic activity and cell proliferation [2,26]. Instead, we

found that blastocyst growth was still active in Msx1/Msx2d/d uteri with increasing blastocyst cell numbers when

compared with blastocysts recovered from Msx1/Msx2f/f

uteri (figure 3b,c). These results provide evidence that blasto-

cyst dormancy is under maternal control, because blastocysts

failed to undergo complete dormancy owing to an altered

uterine environment in Msx1/Msx2d/d females.

To further explain the asynchronous activities between

the blastocyst and uterus in the Msx1/Msx2d/d females, we

examined the status of Wnt5a expression in ovariectomized

P4-treated, floxed and deleted females, because Wnt5a is a

transcriptional target of Msx in the uterus during implan-

tation and is upregulated in Msx-deleted uteri [7,27]. On

day 10 under dormant conditions, Msx1/Msx2d/d uteri

showed higher levels of Wnt5a expression in the epithelium

and subepithelial stroma when compared with Msx1/Msx2f/f uteri under true delay, and Wnt5a expression was

markedly upregulated in the pseudo-implantation sites at

the mesometrial side in Msx1/Msx2d/d uteri (figure 3d ).

Wnt5a expression at these sites reflected the induction of its

expression in activated uteri undergoing implantation after

LIF or E2 administration (see the electronic supplementary

material, figure S3). These results suggest paracrine signalling

by Wnt5a to the blastocyst. Taken together, these findings

provide evidence that Msx1/Msx2d/d uteri fail to achieve

quiescence, leading to discordant blastocyst activation and

ultimately demise of the embryo.

4.3. Oestrogen and P4 are not the primary inducers ofMsx1 in the mouse uterus

The above results raised questions whether Msx genes are regu-

lated by P4 and/or oestrogen, because these hormones are

prime endocrine factors involved in embryonic diapause and

activation. To determine whether Msx1 expression is regulated

by ovarian hormones, ovariectomized Msx1LacZ reporter mice

were treated with vehicle (sesame oil), P4 (2 mg), E2 (25 ng) or

P4þ E2 for 24 h. Surprisingly, Msx1 was distinctly expressed

MSX1MSX1

mink uterus

myo

gele

d0

d9

d14

d14

Emb

Emble

le

myo

le

darkfield brightfieldge

reactivation (d9)

1.1

WNT5A

0.9

0.7attachment

IIS IS

0.5

0.3

0.1

implantation site (d14)

reactivation (d9)diapause implantation (d14)

diap

ause

MSX

1 ex

pres

sion

rela

tive

to G

APD

H

diap

ause

reac

tivat

ion

impl

anta

tion

nuclearmerged

(a)

(b)

(d )

(c)

day of embryonic reactivationdia d5 d14 d14

Figure 4. MSX1 is highly expressed in diapausing mink uterus (Neovison vison) and downregulated upon embryonic reactivation with WNT5A expression in a reciprocalmanner. (a) In situ hybridization results show distinct MSX1 expressed in luminal and glandular epithelia in the diapausing uterus. Its downregulation was seen on day 9 ofreactivation (d9) followed by undetectable levels at the implantation site on day 14 (d14). Arrowheads indicate glands. Scale bar, 500 mm. (b) qPCR results show down-regulation of MSX1 approaching attachment. IIS, interimplantation site; IS, implantation site. (c ) Immunofluorescence shows MSX1 nuclear localization during diapause withreduced signals on day 9 of reactivation and undetectable signal at the implantation site. Scale bar, 100 mm. (d) In situ hybridization results show undetectable WNT5Aexpression in the diapausing uterus, and modest expression in luminal and glandular epithelia on day 9 of reactivation. By day 14, WNT5A is robustly expressed in theluminal epithelium and subepithelial stroma. Scale bar, 500 mm. d0, diapause; Emb, embryo; myo, myometrium; ge, glandular epithelium; le, luminal epithelium.

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in luminal and glandular epithelia in vehicle-treated uteri, and

levels of expression were comparable among different treatment

groups (see the electronic supplementary material, figure S4a).

Msx1 expression persisted in Lif2/2 uteri under similar

treatment, whereas LIF injection in Lif2/2 females induced

implantation with downregulation of uterine Msx1 expression

(see the electronic supplementary material, figure S4b). These

results suggest that regulation of Msx1 is not directly under

the influence of E2 and/or P4 in mice.

We also found that exogenous P4 administration on days

3 and 4 of pregnancy did not rescue implantation failure in

Msx1/Msx2d/d females, although similar P4 treatment did

not interfere with normal implantation in littermate floxed

females (see the electronic supplementary material, figure

S5a,b). This is consistent with comparable P4 responsiveness

in floxed and deleted mice as assessed by expression of two

P4-responsive genes Indian hedgehog (Ihh) and Hoxa10 on

day 4 of pregnancy (see the electronic supplementary

material, figure S5c). By contrast, Ki67 and phospho-histone

immunostaining results suggest that luminal epithelial pro-

liferation persisted in deleted uteri on day 4, as opposed to

little to no signal in the fully differentiated epithelium in

day 4 floxed uteri (see the electronic supplementary material,

figure S5d ). These results suggest that incomplete luminal

epithelial differentiation in the deleted uteri prevented

receptivity towards blastocyst attachment.

4.4. MSX1 is highly expressed during diapause in theNorth American mink

The results in mice described above prompted us to examine

whether MSX genes play roles in other diapausing mammals,

in particular, the American mink (N. vison) and Australian

tammar wallaby (M. eugenii). The American mink is one of

many mustelid carnivores that undergo seasonal obligate

delay, which can persist for more than nine months in some

species [1,4]. Although embryos develop into blastocysts,

their further growth is arrested. Embryo reactivation can be

triggered by prolactin administration with increased P4

output and uterine polyamine synthesis, with implantation

occurring 12 days later [1,28]. We found increased MSX1expression in luminal and glandular epithelia of diapausing

mink with reduced expression on day 9 after embryonic reac-

tivation, followed by undetectable levels at implantation sites

on day 14 (figure 4a). This expression pattern corroborates

diap

ause

(d0

)

diap

ause

(d0

)d9

RPY

d17

RPY

reac

tivat

ion

(d25

)

MSX

2 ex

pres

sion

rela

tive

to A

CT

B

MSX1

se

ge

myo

entry into

d1 p

y

d2 p

y

d3 p

y

d4 p

y

d6 p

y

d0 R

PYd0

RPY

d3 R

PY

d4 R

PY

d5 R

PY

d6 R

PY

d10

RPY

d12

RPY

d15

RPY

d17

RPY

d20

RPY

d25

RPY

diapause

(–)

reactivation from

MSX2

MSX2 MSX2

MSX2 H&E

ACTB

1.2

1.0

0.8

0.6

0.4

0.2

0d4 py d0day of reactivation (in days)

d5 d25 d26

(a) (c) (d)

(b)

Figure 5. MSX2 expression persists in diapausing tammar wallaby uteri (Macropus eugenii) and is progressively downregulated upon embryonic reactivation. (a) Repre-sentative RT-PCR results of MSX1, MSX2 and ACTB at various stages of diapause and reactivation showed that MSX2 expression remains high during diapause (d0,diapause; 68 and 145 days postpartum) with negligible expression of MSX1. Embryo reactivation by RPY showed gradual decreases in uterine MSX2 expression approach-ing embryonic attachment (day 17 RPY) and towards delivery (days 25 – 26 RPY). py, pouch young indicating days postpartum. (b) qPCR results showed heightenedMSX2 expression relative to ACTB at diapause with gradual decreases during reactivation. (c) In situ hybridization results showed sustained MSX2 expression in epitheliaduring diapause with downregulation on day 25 RPY. ge, glandular epithelium; se, surface epithelium; myo, myometrium. (d ) Immunohistochemistry showed distinctnuclear localization of MSX2 in glandular and surface epithelia during diapause with gradual reduction on day 9 of reactivation from the surface epithelium and under-lying glandular epithelium, but expression persisted in the deeper glands (left panels, scale bars, 250 mm; inset in right panels, scale bars, 100 mm). No positive signalwas detected by day 17 of reactivation (bottom left panel). H&E staining on day 17 (bottom right panel). Scale bars, 250 mm.

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qPCR and immunofluorescence results (figure 4b,c). Intrigu-

ingly, uterine WNT5A expression in the mink showed an

inverse relationship with MSX1 expression similar to that

seen in mice: WNT5A expression was undetectable in the dia-

pausing uterus and correlated with higher uterine MSX1expression. By contrast, diminishing expression of MSX1 was

reflected in higher WNT5A expression. It was modestly

expressed in the luminal epithelium and subepithelial stroma

upon embryonic reactivation on day 9 but was robustly

induced in these cell types upon implantation on day 14

with gradual tapering of expression towards the muscular

layers (figure 4d). Hybridization with sense probes for

MSX1 or WNT5A did not show any positive signals (see the

electronic supplementary material, figure S6a,b); MSX2 was

not detected by RT-PCR, although these primers could

detect compensatory Msx2 expression in mouse uteri deleted

of Msx1 (data not shown) [7].

4.5. MSX2 expression persists during diapause in theAustralian tammar wallaby

The tammar wallaby undergoes a period of lactational delay

during the first half of the year (February–June in the Southern

Hemisphere) and a seasonal diapause after the winter solstice

in June, thereby maintaining diapause for 11 months of the

year [3,29,30]. In the tammar, the long duration of diapause,

which can be extended further after the removal of the ovary

[31], requires a supportive uterine environment because its

secretory activity is minimal during diapause [13]. Remarkably,

reactivation occurs synchronously among all pregnant walla-

bies during the summer solstice, the longest day of the year

(22 December) with birth occurring 25–30 days later (approx.

22 January). Mating occurs within 1 h of birth, and the new

conceptus reaches the 80-cell blastocyst stage to enter diapause

[3]. Meanwhile, the newborn young remains in the mother’s

pouch for about nine months for the remainder of development

and its nursing prevents reactivation of the blastocyst formed at

the postpartum mating; RPY allows embryonic reactivation

and attachment. We found that MSX1 was undetectable in

the pregnant uterus throughout diapause. By contrast, RT-

PCR and qPCR results showed that MSX2, which has overlap-

ping function with MSX1, was highly expressed during

diapause with expression progressively diminishing after

embryonic reactivation up to day 5 after RPY, reaching lowest

levels after attachment to the endometrium which normally

occurs on day 18 after RPY (figure 5a,b). Expression was

undetectable approaching parturition on day 25 prior to birth

on day 26.5. Again, both MSX2 mRNA and MSX2 protein

were highly expressed in the glandular and surface epithelia

during diapause (figure 5c,d). Hybridization with a sense

probe did not exhibit positive signal (see the electronic sup-

plementary material, figure S6c). Intriguingly, MSX2

expression upon embryonic reactivation first becomes undetect-

able in the surface epithelium and superficial epithelial glands,

with deeper glands still expressing MSX2 on day 9 of reactiva-

tion with levels becoming undetectable by day 17 (one day

before blastocyst attachment; figure 5d).

5. DiscussionAlthough endocrine directives for embryonic diapause and

reactivation have been described in several mammalian

species [1,2], local molecular mechanisms that instruct the

initiation and maintenance of diapause remain unknown.

Msx genes are highly conserved across Metazoa from

sponges to vertebrates and are considered ancient among

homeobox gene families [8]. Our findings provide the first

evidence linking Msx1 and Msx2 to the regulation of

Figure 6. Potential schematic of the regulation of diapause in mouse, minkand wallaby. Although there is variation in the proximate stimuli used toinduce diapause in these species in relation to differing roles of prolactin(PRL), progesterone (P4) and oestradiol-17b (E2), these diverse pathways ulti-mately converge to work through MSX in the uterus, which controls diapauseand reactivation of the blastocyst, with an inner cell mass in eutherian mam-mals but not in marsupials. Genetic and molecular studies in mice show a lackof true diapause in the absence of uterine Msx genes potentially via upregula-tion of Wnt5a as a paracrine regulator for blastocyst growth, precluding fullyfledged dormancy. Larger font size indicates higher levels of hormones.

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embryonic diapause. Of particular interest is the finding that

the Msx expression pattern is conserved across distantly

related taxa and between facultative (lactational) and obligate

(seasonal) diapause. Although different species use distinct

strategies to confer uterine quiescence synchronized with

embryonic diapause to defer implantation, a recent interspe-

cies embryo transfer study shows that embryos from non-

diapausing sheep can enter diapause when placed in a

delayed implanting mouse uterus and can be reactivated to

produce normal offspring upon transfer back into the

donor uterus [32]. This finding again implicates maternal

regulation of embryonic diapause and suggests that embryos

from non-diapausing species have the capacity to enter

diapause if the maternal environment is conducive to this

event. It further suggests that diapause is an ancient and con-

served reproductive strategy and that all mammals may be

capable to undergo diapause given the appropriate maternal

regulatory signals [32]. Whether humans and their close

primate relatives have or once had the potential to undergo

delay remains uncertain [33]. Interestingly, gene array results

show that MSX1 and MSX2 are expressed in the human

endometrium in a similar manner to that in mice, with

higher expression prior to and downregulation during the

window of implantation (mid-secretory phase) [34,35].

These results may intrigue investigators to explore the

status of uterine Msx expression in other diapausing and

non-diapausing species.

Our studies in mice provide genetic and molecular evi-

dence that Msx genes are critical to the initiation and

maintenance of embryonic diapause, and uterine readiness

to confer blastocyst reactivation and implantation. Increased

Wnt5a expression in Msx1/Msx2d/d uteri may function as a

paracrine signal to promote proliferation of blastocysts

entrapped within the uterine lumen without invasion, leading

to their progressive demise within the hostile environment in

Msx-deleted uteri. This is a potential explanation for the lack

of true blastocyst dormancy in Msx-deficient uteri, because

we have previously shown Wnt5a as a transcriptional target

of uterine Msx [7]. Interestingly, we found increased WNT5Aexpression in the mink uterus after reactivation and implan-

tation with diminishing MSX1 expression similar to the

pattern in mice, suggesting this inverse relationship between

Msx1 and Wnt5a is conserved in the mouse and mink. It

remains to be seen whether WNT5A in the tammar wallaby

endometrium is also inversely expressed with MSX2.

Although P4 can prolong blastocyst survival under delayed

conditions in mice, diapausing blastocysts of all three species

can still survive for many days after ovariectomy without any

ovarian hormone support [22,31,36,37]. Notably, our results

show that Msx1 expression persists even in uteri of ovari-

ectomized mice without any steroid hormone treatment. It

would be interesting to know whether blastocysts survival and

implantation competency is coincident with MSX expression

in ovariectomized pregnant mink and tammar wallaby uteri.

Nonetheless, uteri in these three species require P4 priming

and/or oestrogen for blastocyst reactivation (implantation

competency) and implantation [38].

Msx proteins are known transcriptional repressors in

various developmental processes in association with the

polycomb repressive complex [39,40]. Our findings of pseudo-

implantation with induction of Ptgs2 and Bmp2 at the site of

blastocyst without trophectoderm invasion through the luminal

epithelium in Msx-deleted mouse uteri on day 10 under an

experimentally induced delayed condition indicate a state of

compromised transcriptional repression. This is consistent

with uterine and blastocyst quiescence seen with persistent

uterine expression of Msx1 in mice and mink and MSX2 in

tammar wallaby during diapause. A deeper molecular under-

standing of synchronized uterine quiescence with blastocyst

dormancy regarding potential upstream regulators and down-

stream targets that function through Msx in diapause in

various species will require in-depth investigation. Nonetheless,

our study has identified Msx genes as a common molecular link

across three species representing different orders with diverse

reproductive strategies (figure 6).

6. AcknowledgementsThe authors thank Susanne Tranguch for some in situ hybrid-

ization experiments. We thank members of the tammar

wallaby and mink research groups for help with the animal

handling and field collections. This work was supported in

part by NIH grants (nos. HD12304, HD068524 and

DA06668 to S.K.D.), Australian Research Council grants (to

G.S. and M.B.R.) and a NSERC Canada grant (no. 137013

to B.D.M). J.C. is supported by an NIH NRSA Fellowship

(F30AG040858) and the University of Cincinnati MSTP

(T32 GM063483). X.S. is supported by a Lalor Foundation

Postdoctoral Fellowship.

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